CN115851757A

CN115851757A - Application of AP2/ERF transcription factor OsDREB2B gene

Info

Publication number: CN115851757A
Application number: CN202211005186.0A
Authority: CN
Inventors: 姜文洙; 马梓铭; 吴涛; 都兴林
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2022-08-22
Filing date: 2022-08-22
Publication date: 2023-03-28

Abstract

The invention relates to application of an AP2/ERF transcription factor OsDREB2B gene, belongs to the technical field of biology, and provides a super-expression vector of a rice AP2/ERF transcription factor OsDREB2B gene, which comprises an OsDREB2B gene expression box, a promoter containing a corn ubiquitin gene Ubi, a target gene OsDREB2B, a 3' end untranslated region NOS with a terminator being a nopaline synthetase coding region of agrobacterium, and a screening marker gene expression box, a promoter CaMV35S containing cauliflower mosaic virus, wherein the marker gene is hygromycin phosphotransferase gene HPT and the terminator NOS; the nucleotide sequence of the AP2/ERF transcription factor OsDREB2B gene is shown in SEQ ID No.1, and the gene can regulate the plant height of rice and has obvious effect and value in practical application.

Description

Application of AP2/ERF transcription factor OsDREB2B gene

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of an AP2/ERF transcription factor OsDREB2B gene in rice.

Background

Rice is one of the most important grain crops in the world and plays an important role in ensuring grain safety. The plant height is one of the important factors determining the plant type of rice. The proper plant height can reduce the leaf density, improve the yield and prevent lodging. Plant height is regulated by various hormones, such as Gibberellin (GA), brassinosteroids (BR), indole-3-acetic acid (Aux/IAA), and Strigolactones (SLs). The administration of exogenous GA3 significantly promoted elongation of the second leaf sheath of OsRPH1 overexpressing rice lacking GA content. The BR can obviously increase the leaf included angle of the rice flag leaf, and compared with the wild type, the BR-insensitive OsOFP22 overexpression rice flag leaf has obviously reduced leaf included angle. Aux/IAA regulates phototropism/targetry, root formation, apical dominance, stem/hypocotyl elongation and leaf expansion. Overexpression of OsIAA1 changed leaf angle, while overexpression of OsIAA4 changed tiller angle.

Gibberellins are one of the most important hormones affecting strains. To date, a total of 136 gibberellins have been identified, of which GA1, GA3, GA4, and GA7 have been shown to be endogenous bioactive gibberellins in plants, some with at least some inherent biological activity, but which are not present in vegetative tissues and thus may not have regulatory functions. Many rice GA mutants are deficient in the GA biosynthetic pathway or in GA signal perception, including GA-deficient mutants and GA-insensitive mutants. GA-deficient mutations are caused by mutations involved in the GA biosynthetic pathway or GA-inactivating enzymes, rather than mutations in the GA signaling gene, and thus plant height can be restored to WT levels under conditions in which GA3 is applied externally. GA-insensitive mutants are caused by mutations in genes involved in GA signaling, and the endogenous bioactive gibberellin content is usually much higher than WT.

In rice, GA-metabolizing genes including catalytic enzymes in the early steps of GA biosynthesis, such as CPS, KS, KO and KAO, are encoded by a single gene. Genes later in GA biosynthesis, such as GA20ox, GA3ox and GA2ox, are encoded by a multigene family. Among them, GA20ox and GA3ox convert GA precursors to bioactive gibberellins through a catalytic oxidation cascade, whereas GA2ox is important for GA inactivation. In the GA signaling pathway in rice, GID1 encodes a soluble GA receptor, and GID2 is the F-box subunit of Skp1-Cullin-F box protein (SCF) E3 ubiquitin ligase, promoting the degradation of rice SLR1 by 26S proteasome in the presence of GA.

The AP2/ERF family is a transcription factor which is specific to plants and plays an important role in the growth and development of the plants and the response process of abiotic stress, such as growth and development and abiotic stress response. In addition, many AP2/ERF family transcription factors, such as OsEATB, osRPH1 and OsAP2-39, influence the GA metabolism level by regulating the transcription expression of GA metabolism genes, thereby regulating the growth and development of plants. Many DREB subgroup genes have been reported to influence abscisic acid (ABA) biosynthesis and signaling to regulate both biotic and abiotic stress response signal transduction pathways, such as OsDREB2A, osDREB1F, osDREB6, and OsARAG1.

The OsDREB2B gene belongs to a DREB subgroup, and researches show that the OsDREB2B gene responds to abiotic stress such as drought, heat stress, low-temperature stress and the like in arabidopsis thaliana or rice. Interestingly, there is a correlation between low temperature stress and GA metabolism. For example, under low temperature conditions, the mRNA expression level of OsDREB2B in rice is up-regulated, while the mRNA expression levels of GA biosynthetic genes GA20ox3 and GA3ox1 are down-regulated. Research shows that the DREB family gene SlDREB in the tomato can reduce the expression of GA related genes, and the overexpression of the gene SlDREB family gene in transgenic tomatoes can cause dwarfing and shorten internode elongation, however, the research of OsDREB2B on the aspects of rice growth and development regulation is not clear.

Disclosure of Invention

The invention aims to provide an AP2/ERF transcription factor OsDREB2B gene, the nucleotide sequence of which is shown in SEQ ID No.1, and the gene can be applied to the regulation of the plant height of rice.

The invention also aims to provide a super-expression vector of the rice AP2/ERF transcription factor OsDREB2B gene, which comprises an OsDREB2B gene expression box, a promoter containing a maize ubiquitin gene Ubi, a target gene OsDREB2B, a non-translated region NOS at the 3' end of a terminator, which is a nopaline synthetase coding region of agrobacterium, and a screening marker gene expression box, which contains a cauliflower mosaic virus promoter CaMV35S, wherein the marker gene is hygromycin phosphotransferase gene HPT and the terminator NOS.

The invention also aims to provide a recombinant cell of an overexpression vector containing the rice AP2/ERF transcription factor OsDREB2B gene.

The invention also aims to provide a primer for detecting the OsDREB2B.

The invention mainly researches the regulation and control mechanism of the OsDREB2B on the growth and development of plants. The OsDREB2B overexpression rice plant has the phenotype of short plant height, short internode length and small seed size and thickness. The invention researches the GA-mediated physiological process of second leaf sheath extension, various endogenous GA contents and the change of the transcription level expression quantity of GA related genes in an OsDREB2B overexpression rice strain and a knockout strain. In addition, downstream target genes and interacting proteins of OsDREB2B have been elucidated. OsDREB2B interacts with OsWRKY21 protein and binds to OsAP2-39 promoter region. Research shows that both OsAP2-39 and OSWRKY11 can regulate the growth of rice. The results of the invention show that: osDREB2B plays a role in negative regulation in the growth and development of rice by regulating the expression of GA metabolic genes, and the expression of the GA metabolic genes is mediated by OsAP2-39 and OsWRKY21, so that the GA content is reduced, and the dwarfing of the plant height of the rice is caused.

The invention has the beneficial effects that: the OsDREB2B can adjust the plant height of rice and has obvious value in practical application. And the over-expression of the gene can be used for carrying out crop genetic improvement and cultivating the semi-dwarf transgenic crop.

Drawings

FIG. 1 is a schematic representation of the phenotype of over-expressed (OE) and wild-type (WT) rice plants;

wherein: (A, B) WT of the whole plant morphology and seedling stage of the OE line. Bar =2cm. (C, D) the whole plant morphology of OsDREB2B-OE and WT in the mature period. Bar =10cm. (E) Cone inflorescences and internodes of OE-1 (right) and WT (left) main culms. Bar =5cm. (F) Internode length and weight (G) seed phenotype of the OE line. Bar =1cm. (H) seed size of OE and WT;

FIG. 2 is a schematic representation of the expression pattern of OsDREB2B in rice;

wherein: (A) Transcript levels of OsDREB2B in various tissues and organs were examined by qRT-PCR. The transcription level of young ears was set to 1. Data are mean ± SD (n = 3). (B, C, D) the effect of different concentrations of GA3 administration on OSDREB2B expression profiles was analyzed by qRT-PCR. Student t-test was used to generate P-values-there was a significant difference between WT and OE when P = 0.01;

FIG. 3 is a schematic representation of the dwarfing phenotype of OE plants that can be restored with GA 3;

wherein: 10 day old OE and WT seedlings were treated with 0, 10, 50 and 100 μ M GA3. At least 10 rice seedlings were measured per line. Data are presented as mean (± SD) of 10 samples per genotype;

FIG. 4 is a schematic representation of the determination of the endogenous GA content in seedlings of WT and OE lines;

wherein: (A) Comparison of major Gibberellin (GA) biosynthetic pathways in plants. (B) GA12 pathway and GA53 pathway. FW, fresh weight. Student t-test was used to determine statistical significance. FW, fresh weight; error bars represent ± SE (n = 3). Student t-test was used to generate P-values-there was a significant difference between WT and OE when P = 0.01;

FIG. 5 is a schematic diagram of expression analysis of GA metabolic genes (A, B, C) and a signaling gene (D);

wherein: the transcription level of WT was set to 1. Data are mean ± SD (n = 3). Student t-test was used to generate P-values-there was a significant difference between WT and OE when P = 0.01;

FIG. 6 is a schematic representation of the transactivation activity and subcellular localization of OsDREB 2B;

wherein: (A) transactivation assay of OsDREB2B protein in yeast. BD. GAL4-DNA binding domain. The OsRPH1 protein was used as a positive control. (B) And the subcellular localization of the OsDREB2B-GFP fusion protein in the rice protoplast cells. D53-RFP was used as a nuclear marker. Bar =10 μm;

FIG. 7 is a schematic representation of OsDRE 2B's direct regulation of OsAP 2-39;

wherein: (A) relative expression of the OsAP2-39 gene in WT and OE lines. (B) combination of OsDREB2B and OsAP2-39 in yeast. GAD-OsDREB2B activates expression of LacZ reporter gene in yeast driven by the OsAP2-39 promoter. GAD and GAL4 are transcriptional activation domains. (C) schematic representation of vectors in a dual luciferase reporter system. (D) in vivo dual luciferase reporter assay. Flag-OsDREB2B and OsAP2-39pro were co-transformed into rice protoplasts with. The FLUC and RLUC activities were measured and Renilla Luciferase (RLUC) was used as an internal control. Student t-test used to generate P-values there was a significant difference between WT and OE at P = 0.01;

FIG. 8 is a schematic representation of OsDREB2B interaction with OsWRKY21 in yeast and plant cells;

wherein: (A) Detecting the interaction of OsDREB2B and OsWRKY21 in the yeast cell; AD, activation domain; BD. A GAL4-DNA binding domain; (B) The BiFC is used for detecting the interaction between OsDREB2B and OsWRKY21 in the rice protoplast; bright, bright areas; YFP, YFP fluorescence; labeling, labeling fluorescence; bar =10 μm;

FIG. 9 is a schematic diagram of a mechanism model of OsDREB2B regulating rice plant height;

wherein: spindles represent different transcription factors and circles represent different suppressors. Rectangles represent cis-element binding sequences; arrows indicate that the OsDREB2B/WRKY21 complex can be directly combined with cis-elements on a GA metabolic gene promoter. The black arrows indicate activation effects and the hammer lines indicate suppression effects. The curved arrows in the dark indicate the transcriptional expression of the genes, and the thickness of the lines indicates the relative transcriptional level.

FIG. 10 is a schematic representation of the development of OsDREB2B overexpressing transgenic rice;

wherein: and (A) a schematic diagram of an OsDREB2B plant expression vector. OsDREB2B is expressed under the control of a maize ubiquitin (Ubi) promoter, and the hygromycin (hgy) gene is expressed under the control of a 35S promoter as a selectable marker in rice. (B) And carrying out PCR analysis on the T0 transgenic rice by using the constructed specific primer. M, marking; WT, kitaake;1-18, independent transformants at passage T0. (C) The T3 generation of the OsDREB2B-OE strain was confirmed by genomic DNAPCR analysis. (D) OsDREB2B expression was analyzed by qRT-PCR in WT and OE. Data are expressed as mean ± SE values from two independent experiments (n = 3). * This indicates that P.ltoreq.0.01 compared to WT in the student sample t test.

FIG. 11 is a schematic diagram of the main stem length comparison of OE-2 and WT.

FIG. 12 is a schematic diagram of the construction of Osdreb2b knockout mutants of rice;

wherein: (A, B, C) Osdreb2B mutants were generated in rice using a set of regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas 9) systems. (D) OsDREB2B expression was analyzed by qRT-PCR in WT and mt. Data are expressed as mean ± SE values from two independent experiments (n = 3). * It indicates that P.ltoreq.0.01 compared to WT in the student sample t test.

FIG. 13 is a schematic representation of the morphology of WT and Osdreb2b mutants;

wherein; (A) Whole plant morphology of WT and Osdreb2b mutants at seedling stage. Bar =1cm. (B) second leaf sheath of WT and Osdreb2B mutants. (C) kernel morphology. Bar =1cm. (D) WT and Osdreb2b mutant sizes. * Indicates that P.ltoreq.0.01 compared to WT in student sample t-test.

FIG. 14 is a schematic diagram showing the measurement of endogenous bioactive gibberellins (GA 1, GA3, GA 4) in WT and Osdreb2b mutant seedlings;

wherein; three biological replicates and three technical replicates were performed on each sample and statistical significance was determined using student sample t-test. FW, fresh weight; error bars represent ± SE (n = 3).

FIG. 15 is a schematic representation of a yeast self-activation inhibition assay;

wherein; (A) pBridge-OsDREB2B and pBridge are reconstructed respectively. Yeast single-hybrid analysis of the indicator proteins was performed on minimal synthetically defined media containing SD/-Trp-Leu-Ade-His conjugate supplements AT different concentrations of 3-AT. (B) Three truncated bait carriers of OsDREB2B, pGBKT7-OsDREB2B-F, pGBKT7-OsDREB2B-N and PGBKC7-OsDREB2B-C respectively represent the truncated structures of the OsDREB2B full length, the N end and the C end of the inserted PGBKT 7. The indicated proteins were tested on minimal synthetic defined media containing SD/-Trp, SD/-Trp-His, SD/-Trp-Ade-His, and SD/- -Trp-Leu-Ade His + X-. Alpha. -gal droplets.

FIG. 16 is a schematic diagram of an X- α -gal color reaction in yeast;

wherein: positive and negative controls correspond to + and sign. Yeast cells co-transformed with pGBKT7-53 (+) and pGADT7 were used as positive controls, and pGBKT7-Lam (-) and PGADT7 were co-transformed as negative controls. The indicated proteins were cultured on minimal synthesis defined medium containing SD/-Trp-Leu, SD/-Trp-Leu-His, SD/-Trp-Leu-Ade-His, and SD/-Trp-LeuAde-His + X-. Alpha. -gal droplets.

Detailed Description

The invention is described below with reference to the drawings.

1. Materials and methods

1.1 plant Material

The full-length CDS of OsDREB2B was cloned and fused to the overexpression vector pCUbi1390, which was driven by the maize ubiquitin promoter. The vector pUbi was constructed by PstI and HindIII sites: : osDREB2B. pUbi: : osDREB2B is transformed into japonica rice Kitaake (Nishimura et al, 2006) through an agrobacterium tumefaciens (EHA 105-) mediated genetic transformation method, and the specific method comprises the following steps: after the rice seeds with glumes removed are disinfected, callus is induced and subcultured, and the rice seeds containing pUbi: : and infecting callus with agrobacterium strain EHA105 bacterial liquid of OsDREB2B carrier, screening hygromycin resistance, differentiating, rooting, and finally transplanting to obtain transgenic independent transformed plant. And obtaining the T3 homozygous line from the progeny plants through molecular detection and hygromycin resistance screening.

1.2 Construction and identification of osdreb2b mutant

Osdreb2b mutants were obtained using CRISPR/Cas9 genome editing techniques. Cas9/gRNA target selection and vector construction reference predecessor studies (Li et al, 2017). The OSDREB2B-pBGK032 vector was introduced into Agrobacterium EHA105 and transformed into Kitaake (Nishimura et al, 2006), as described above. And (3) screening OSDREB2B homozygous mutants by analyzing mutation conditions through PCR detection and DNA sequencing of OSDREB2B specific editing sites.

1.3 phenotypic analysis, statistical analysis and growth conditions

The rice plants are planted in the agriculture experiment base of Jilin university in Changchun city, jilin province, and the normal cultivation condition is managed. The OE and mt lines were hydroponically grown for 14 days under alternating dark (30 ℃,12h light/24 ℃,12h dark) conditions and then evaluated for seedling phenotype. Transplanting the seedlings to the experimental field in the rice growing season. After the mature seeds were air dried, the seed length, width and thickness were measured. 10 plants were used for phenotypic analysis using student t-test.

1.4 GA3 treatment

WT seeds were germinated in distilled water and grown in kimura B broth. After 14 days of culture, seedlings were treated with 10, 50 and 100 μ M GA3 (Sigma-Aldrich, china, shanghai) for three biological replicates. Seedlings were collected at 0, 1, 6 and 12 hours after treatment. In the recovery experiment of externally applied GA3 (Ma et al, 2020), WT and transgenic rice seedlings in the trefoil stage were cultured in kimura B culture medium for 10 days, and then GA3 was sprayed at concentrations of 10, 50 and 100. Mu.M, respectively. The length of the second leaf sheath, 20 strains/replicate, three biological replicates, was measured every 6 hours. Values are mean ± sd.

1.5 determination of endogenous gibberellin content

Endogenous GA content of WT, OE and mt strains was determined by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). 1g of shoot tissue was collected from 10-day-old rice seedlings, and the content of endogenous gibberellin was measured by the method of Li et al (2017). Three technical replicates were performed for each sample and three biological replicates were performed and statistical significance was determined using student t-test.

1.6 RNA extraction and real-time quantitative PCR analysis

Total RNA was extracted from leaves of 14-day-old rice seedlings using an RNA extraction kit (tiangen biotechnology limited, china, beijing), and reverse transcription was performed using a superscript II kit (TaKaRa, japan, tokyo). Real-time PCR was performed on an ABI one-step PCR system (applied biosystems, foster, ca, usa) using an SGExcel FastSYBR qPCR cocktail (bio-technologies, ltd, china, shanghai). Each reaction contained 10. Mu.L of SGexcel FastSYBR qPCR mix, 0.2. Mu.M primer, and 1. Mu.L of template cDNA. The PCR reaction parameters were 95 ℃ for 2min (1 cycle), 95 ℃ for 15S and 60 ℃ for 20S (40 cycles), and then melting curve analysis was performed at 95 ℃ for 60S, 55 ℃ for 30S and 95 ℃ for 30S. The rice OsUbiquitin and OsActin genes were used as internal reference genes (Zheng et al, 2015 et al, 2019. Three technical replicates and three biological replicates were performed per qRT-PCR analysis. .

1.7 Yeast transactivation Activity assay

The full-length Open Reading Frame (ORF) of OsDREB2B was inserted into pBridge vector to construct pBridge-OsDREB2B. pBridge-OsRPH1 was used as a positive control and the empty pBridge vector was used as a negative control (Ma et al, 2020), transformed into yeast strain AH109 (Clontech). Positive transformants were verified on a triple-gap medium (SD/-Trp-Ade-His) and dropped in a quadruple-gap medium (SD/-Trp-Leu-Ade-His).

1.8 Subcellular localization of OsDREB2B

The coding region of OsDREB2B is connected to pAN580 GFP vector driven by CaMV35S promoter to construct OsDREB2B-GFP vector. The D53-RFP fusion protein was used as a nuclear marker (Zhou et al, 2013). Rice protoplasts were prepared and transformed using the method of Wang et al (2016). The transient expression vector is transformed into rice protoplast cells and cultured under dark conditions at 28 ℃ for 12-14 hours. Fluorescence images were observed using a confocal laser microscope (Carl Zeiss, aJena, germany).

1.9 CDNA library construction

A rice cDNA library was constructed by extracting total RNA from 2-week-old rice seedlings. First strand cDNA was synthesized using SMARTM cDNA library construction kit (Clontech) with 1. Mu.g of total RNA as a template by means of oligonucleotide (dT) primer. Double-stranded cDNA was synthesized by long-range PCR (LD PCR). After quality check, dscDNA was inserted into pGADT7SfiI vector after SfiI digestion and then transformed into e. Six single positive colonies grown on LB medium (Amp 100 g/L) were randomly selected and PCR amplified with pGADT7 primers: the forward primer is 5 'TAATACGACTAGG-containing 3'; the reverse primer is 5 'and GGCAAACGATGTATAATGA-3'. pGBKT7-53 (+) and pGADT7 were co-transformed yeast as a positive control (-), pGBKT7-Lam was co-transformed yeast as a positive control (-), and pGADT7 was used as a negative control (Chen et al, 2017). The above vector was transformed into yeast strain AH109 (Clontech) and positive transformants were verified on four-way medium (SD/-Trp-Leu-Ade-His) and dropped into X-gal-added four-way medium (SD/-Trp-Leu-Ad-His + X-gal).

1.10 Yeast two-hybrid (Y2H) assay

The cDNA fragment coding the OsDREB2B is amplified and cloned into a pGBKT7 vector, and a gateway system is used for constructing a 'bait' vector pGBKT7-OsDREB2B. Y2H screening was performed using cDNA libraries prepared from rice seedlings and positive clones were identified by DNA sequencing. The coding region of OsWRKY21 was inserted into pGADT7 vector as "prey". The "bait" and "prey" constructs were co-transformed into yeast strain AH109 (Clontech). Positive transformants were verified on three-medium (SD/-Trp-Leu-His) and transferred to four-medium (SD/-Trp-Leu-Ade-His). All steps were performed according to the manufacturer's user manual (Clontech Laboratories).

1.11 bimolecular fluorescence complementation (BiFC) assay

The cDNA fragment encoding OsWRKY21 was amplified and cloned into pXY105 vector carrying YFP (nYFP) -N-terminal half under the control of CaMV35S promoter to form nYFP-OsWRKY21, and the cDNA encoding OsDREB2B was cloned into pXY103 vector carrying YFP-C-terminal half (cYFP) under the control of CaMV35S activator to construct cYFP-OsDREB2B. And co-transferring nYFP-OsWRKY21 and cYFP-OsDREB2B into the rice protoplast. D53-RFP fusion proteins were used as nuclear markers. The transformed rice protoplasts were cultured in the dark at 23 ℃ for 12-14 hours. Fluorescence images were observed using Axio Observer D1 (Carl Zeiss, jena, germany).

1.12 Yeast Single-hybrid (Y1H) assay

The full-length CDS of OsDREB2B is amplified and cloned into a pJG4-5 vector to construct pJG5-OsDREB2B, and an OsAP2-39 promoter (-1500 bp) is cloned into a report vector pLacZi-2 mu. pJG4-5-OsDREB2B and OsAP 2-39pro. The transformants were cultured on SD/Trp Ura plates at 30 ℃ for 3-4 days. DNA sequencing was used to identify putative positive clones. Individual positive colonies were streaked on SD/-Trp-Ura chromogenic plates and cultured at 30 ℃ for 3 days (Lin et al, 2007).

1.13 Dual luciferase reporter assay

The OsAP2-39 promoter (. About.1500 bp) was inserted into the luciferase reporter (LUC) -containing vector pGreen II 0800 to generate a reporter vector and the CDS of OsDREB2B was inserted into a marker vector to generate an effector vector. Subsequently, the reporter vector was co-transformed with an empty marker vector (negative control) or an effector vector (Flag-OsDREB 2B) into rice protoplasts by PEG (Yoo et al, 2007). After 12-14 hours of incubation at 28 ℃, the levels of LUC and reniral-siferase (REN) activities of rice protoplasts were measured using a dual-luciferase reporter assay system (E1910, promega). Transient expression of LUC driven by the OsAP2-39 promoter was normalized to expression of an internal control Reporter (REN).

2 results and analysis

2.1 overexpression of OsDREB2B in Rice has dwarf phenotype

In the earlier stage work, the inventor clones a plurality of transcription factor genes (pUBI: osTFs), transforms the transcription factor genes into a receptor rice variety 'Kitaake' and constructs a transcription factor over-expression (TF-OE) rice library. Through phenotypic evaluation, OE lines with dwarf phenotypes were screened from TF-OE rice libraries. They were found by sequencing to be over-expressed lines of the OsDREB2B (LOC _ Os05g 27930) gene. 14 independent T0 generation OsDREB2B-OE strains are obtained, and the mRNA expression quantity of the target gene is detected. Finally, two OsDREB2B gene overexpression transgenic strains (OE-1 and OE-2) T3 homozygous lines are screened and used as follow-up research materials (figure 10). Both OE lines showed dwarf phenotypes, such as plant height, internode length and thickness, particle size and yield were significantly reduced (fig. 1 and 11). During the seedling stage, the average lengths of the secondary sheaths of OE-1 (3.07 cm) and OE-2 (2.95 cm) were only 70.9% and 68.1%, respectively, of wild type (4.33 cm) (FIGS. 1A and B). At maturity, the average plant heights of OE-1 (68.76 cm) and OE-2 (64.16 cm) were only 81.3% and 75.9% of wild-type (84.56 cm), respectively (FIGS. 1C and D). Comparative analysis of internode length between OE strain and wild type showed that four internodes (first, second, third and fourth) in OE strain were significantly shorter than wild type (E and F of fig. 1).

To further explore the role of OsDREB2B in plant growth regulation, the inventors edited the OsDREB2B gene in the genome of the wild-type rice variety kitaake using the CRISPR/Cas9 system to obtain an OsDREB2B knockout mutant (OsDREB 2B) (fig. 12). Two independent homozygous lines (mt-1 and mt-2) for OsDREB2B knockout were selected by DNA sequencing and molecular detection (B and C of FIG. 12). The second leaf sheath length of mt-1 and mt-2 seedlings was similar to the WT length of 10-day old seedlings (A and B of FIG. 13). mt-1 and mt-2 seeds are similar in length, width and thickness to WT (P.ltoreq.0.01) (C and D of FIG. 13). These results indicate that OsDREB2B has a negative regulatory effect on rice plant height.

2.2 Tissue-specific and GA-responsive expression profiles of OsDREB2

Studies have shown that OsDREB2B is an AP2/ERF transcription factor that is induced under adverse conditions, such as low temperature stress in Arabidopsis (Sakata et al, 2014), drought, salt and heat (Matsukura et al, 2010). The overexpression OsDREB2B effectively improves the tolerance of rice to drought stress (Chen et al, 2008) and the tolerance of arabidopsis thaliana to high temperature and drought stress (Matsukura et al, 2010). RNA sequence database (https:// genev estimator. Com) also confirmed that OsDREB2B expression level of rice is significantly increased under low temperature stress, drought, salt and high temperature stress (data not shown). According to the research results, osDREB2B is presumed to be related to the growth and development of plants as well as abiotic stress. Therefore, the inventors focused on a novel function of OsDREB2B in rice growth, and conducted intensive studies to elucidate the mechanism thereof.

In this study, tissue-specific and GA-regulated expression patterns of OsDREB2B were examined using qRT-PCR. As shown in a of fig. 2, transcripts of OsDREB2B were expressed in many organs, such as leaf sheath, young leaf, stem, flag leaf, young ear, and ear. It is noteworthy that it is significantly highly expressed in the leaf sheath, reaching at least eight times that of the young ear, while the expression level in other organs is very low (a of fig. 2). GA is a hormone for promoting the growth and development of rice, and mainly regulates the elongation of leaf sheath and internode. To analyze GA response expression of OsDREB2B, qRT-PCR was performed on WT rice treated with different concentrations of exogenous GA3. With increasing GA3 concentration, the transcript level of OsDREB2B increased: at 100. Mu.M, it increased rapidly to 2.9-fold within 1 hour (B-D of FIG. 2). Therefore, osDREB2B can be speculated to be involved in GA-regulated biological processes, possibly in plant growth and development.

2.3 application of exogenous GA3 to Rice restores dwarf phenotype of OsDREB2B seedlings

Rice with low endogenous gibberellin content exhibits a dwarfing phenotype, and the short length of the secondary sheath is restored by exogenous gibberellin administration (Spielmeyer et al, 2002; ma et al, 2020). In order to elucidate whether the dwarfing phenotype of the overexpression OsDREB2B line is caused by the reduction of the content of endogenous GA, the seedling growth conditions of OE lines and WT rice under the treatment of exogenous GA3 were studied. 10-day-old seedlings were treated with different concentrations of GA3 (0, 10, 50 and 100. Mu.M), and the elongation of the second leaf sheath was observed at different time points after treatment (A-D in FIG. 3). Elongation of the second leaf sheath increased with increasing GA3 concentration, elongation of the OE strain was faster than WT. After treatment with 100. Mu.M GA3, the length of the second leaf sheath of the OE line was almost the same as that of the WT plants within 48 hours after treatment (FIG. 3D). In addition, exogenous GA3 was also applied to mt lines, but with no significant difference from WT (data not shown). Therefore, exogenous GA3 administration allowed complete restoration of the length of the second leaf sheath of the OE line to WT levels, suggesting that OE line dwarfing was not caused by blocking of the GA signaling pathway, but rather by low endogenous GA content.

2.4 reduction of bioactive GA content in Rice

To confirm the inventors' hypothesis, the inventors analyzed endogenous GA content in OE strains and WT using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). A total of 15 GA contents and distribution patterns were analyzed, including the active state and its metabolites and precursor products (a of fig. 4). Comparing the GA content between OE and WT, in the GA12 pathway, the content of four GAs, i.e. bioactive GA4 and its precursors (GA 15, GA24 and GA 9) decreased, whereas the content of GA51 (catabolite of GA 9) increased, and no GA12 (initial precursor of bioactive GA 4) and no GA34 (metabolite of GA 4) were detected. In the GA53 pathway, the content of five GAs (including bioactive GA1 and GA 3) and their precursors (GA 44, GA19 and GA 20) was reduced; however, the content of GA53, the first precursor of bioactive GA1, and the two catabolites GA29 and GA8 increased (A and B of FIG. 4).

Remarkably, the contents of the three bioactive gases (GA 1, GA3 and GA 4) were significantly reduced (P.ltoreq.0.01), by 62.5%, 75% and 68.7%, respectively (B of FIG. 4); however, no significant difference was observed between mutant lines (mt-1 and mt-2) and WT (FIG. 14). Overexpression of OsDREB2B causes the content of endogenous bioactive GA to be reduced, but inhibition expression of OsDREB2B has no influence on the change of the GA content, which indicates that OsDREB2B negatively regulates the GA content.

2.5 OsDREB2B down-regulates GA biosynthetic genes, but up-regulates GA inactive genes

To confirm OsDREB2B as a negative regulator of GA content, the transcriptional levels of catalytic enzymes at most early and late steps of GA biosynthesis, as well as GA signaling factors, were examined by qRT-PCR in 10-day-old OE lines and WT rice seedlings. Interestingly, in the OE strain, expression of OsCPS1, osKS, osKO2 and OsKAO genes in the early steps of GA biosynthesis were significantly down-regulated (P.ltoreq.0.01), with reductions of 44.4%, 51.8%, 55.5% and 83.7%, respectively, compared to WT (FIG. 4A and FIG. 5A). Similarly, the expression levels of late GA biosynthetic genes, including OsGA20ox1-OsGA20ox2 and OsGA3ox1-OsGA3ox2, were significantly down-regulated in OE (P.ltoreq.0.01) (FIGS. 4A and 5B). In contrast, the expression levels of GA inactivation-associated genes, such as OsEUI (A of FIG. 5), osGA2ox1, osGA2ox3-6, osGA2ox8 and OsGA2ox9, were significantly up-regulated in OE (P.ltoreq.0.01) compared to WT (A and 5C of FIG. 4). Furthermore, the transcripts of the GA signaling factors OsGID1, osGID2 and OsSPY in the OE line did not show significantly differential expression compared to wild type (D of fig. 5), which means that the GA signaling pathway was not blocked by OsDREB2B. This is consistent with the short leaf sheath recovery phenomenon after exogenous GA3 treatment (fig. 3). Taken together, osDREB2B down-regulates bioactive GA content by down-regulating GA biosynthesis genes and up-regulating GA inactivation genes, rather than blocking GA signaling factors.

2.6 OsDREB2B functions as a transcriptional activator in rice

Transcription factors regulate the expression level of downstream genes by binding cis-elements on nuclear genomic DNA sequences. To determine whether the OsDREB2B protein has transactivation activity, osDREB2B was fused to a GAL4-DNA binding domain in pBridge and expressed in yeast. Transformants of pBridge-OsDREB2B and the positive control pBridge-OsRPH1 grew well on four-deficient medium (SD/-Trp-Leu-Ade-His), while the negative control pBridge failed to grow (A of FIG. 6). In the absence of the GAL4 activation domain of pBridge, osDREB2B fuses to the GAL4 DNA binding domain, effectively activating transcription in yeast cells. In addition, the pBridge-OsDREB2B transformants were transferred to a four-deficient medium (SD/-Trp-Leu-Ade-His) containing different concentrations of 3-amino-1, 2, 4-triazole (3-AT) and cultured AT 30 ℃ for 3 days. In contrast to the negative control, pBridge-OsDREB2B still grew normally on 75mM 3-AT (A of FIG. 15), indicating that OsDREB2B acts as a transcriptional activator in yeast.

In order to verify the subcellular localization pattern of OsDREB2B, osDREB2B-GFP fusion proteins expressed under the control of CAMV35S promoter (p 35S:: osDREB 2B-GFP) were constructed and transiently expressed in rice protoplasts. As shown in B of FIG. 6, the control smGFP was uniformly distributed throughout the cytoplasm, whereas the OsDREB2B-GFP fusion protein was localized only in the nucleus. From this, it is inferred that OsDREB2B functions as a transcription activator in rice.

2.7 OsDREB2B regulates and controls transcription level expression quantity through direct combination with OsAP2-39 promoter

Overexpression of the AP2/ERF family transcription factor gene OsAP2-39 makes it possible to reduce the plant height of transgenic rice by up-regulating the GA catabolic gene OsEUI (Elongated Uppermost Internode), which encodes cytochrome P450 monooxygenase, and to inactivate GA by epoxidation (Ma et al, zhu et al, 2006; zhang et al, 2008). Compared to WT, the transcriptional expression level of OsAP2-39 was significantly up-regulated in OsDREB2B overexpression lines (a of fig. 7). Analysis of the promoter results using PlantCARE (http:// bioinformatics. Psb. Element. Be/western tools/planta/html /) showed that the OsAP2-39 promoter region had DRE/CRT cis elements, however, osEUI did not have DRE/CRT or GCC box in the promoter region. Therefore, the inventors speculate that OsDREB2B binds directly to the promoter of OsAP2-39 and activates its expression, excluding the possibility that OsEUI may be a direct downstream target of OsDREB2B.

To confirm the in vivo binding of OsDREB2B to the OsAP2-39 promoter, yeast single-hybrid (Y1H) and dual-luciferase assay experiments were performed. In the Y1H assay, the full-length OsDREB2B fusion protein was fused to GAL4 transcriptional activation domain (GAD), and the OsAP2-39 promoter was fused to LacZ reporter gene. When GAD-OsDREB2B and OsAP 2-39pro. In the dual luciferase assay, the OsAP2-39 promoter was fused to a luciferin protease (LUC) reporter gene and OsDREB2B was driven by 35S as an effector (C of fig. 7). The relative FLuc/RLuc activity ratio in Flag-OsDREB 2B-carrying rice protoplasts was significantly increased compared to when the spap 2-39pro co-transformed with FLuc (fig. 7D). These results show that OsDREB2B directly binds to the promoter of OsAP2-39 and activates its expression.

2.8 OsDREB2B and OsWRKY21 interaction

Some AP2/ERF transcription factors interact with various proteins to exert redundant functions. To identify potential interacting proteins of OsDREB2B, yeast two-hybrid (Y2H) screening was performed using a rice cDNA library. The self-activation activity verification of three truncated bait vectors (full length, N end and C end of OsDREB 2B) shows that the self-activation activity of OsDREB2B needs the C end. Therefore, the truncated OsDREB2B-N without C-terminus was used for screening (B of fig. 15).

In total, 49 proteins were screened as potential proteins for interaction with OsDREB 2B-N. Most proteins grew normally on medium four starvation (SD/-Trp-Leu-Ade-His), 7 of which had a dark blue response (FIG. 16). Among them, a protein OsWRKY21 involved in growth and development of rice was identified as 7 in FIG. 16). The over-expressed OsWRKY21 rice has a semi-dwarf phenotype, an early heading stage and a short internode (Wei et al, 2021), which is very consistent with the phenotype of the over-expressed OsDREB2B strain of the inventor (figure 1).

To further validate the physical interaction between OsDREB2B and OsWRKY21, a one-to-one Y2H analysis was performed. As shown in A of FIG. 8, osDREB2B-pGBKT7 and AD-OsWRKY21 interact when co-transformed into yeast. In addition, bimolecular fluorescence complementation (BiFC) analysis in rice protoplasts also confirmed the interaction between OsDREB2B and OsWRKY 21. Interestingly, when OsDREB2B-nYFP and OsWRKY21-cYFP were co-transformed, YFP fluorescence signal was clearly detected in the nucleus of protoplasts, whereas no signal was detected in the co-transformation reaction of OsDREB2-nYFP and cYFP or OsWRKY11-cYFP and nYFP (FIG. 8B). Therefore, osDREB2B physically interacts with OsWRKY21 in the nuclei of yeast and rice protoplasts.

Discussion of 3

The AP2/ERF transcription factor OsDREB2B belongs to the DREB subgroup, which is reported to cope with abiotic stresses, such as drought, heat and low temperature stresses in arabidopsis or rice. However, little is known about the molecular mechanism of OsDREB2B in rice growth and development. In the research, osDREB2B is used as a dual regulation factor of GA metabolic gene expression mediated by OsAP2-39 and OsWRKY21, and the GA content is reduced, so that the plant height of rice is reduced.

GA is a hormone for promoting the growth and development of rice, and mainly regulates the elongation of leaf sheath and internode. In the inventors' studies, OE lines showed a dwarf phenotype, mainly manifested by leaf sheath shortening and internode length shortening (fig. 1 and 11), while mt lines showed a phenotype similar to WT (fig. 13). These observations indicate that overexpression of OsDREB2B interferes with many aspects of plant growth and development, while knockout lines have no effect on rice growth due to gene redundancy.

Furthermore, exogenous GA3 administration significantly promoted elongation of rice leaf sheaths and affected OsDREB2B transcription (fig. 3). This phenomenon is similar to GA-deficient mutants. Furthermore, the expression of many GA metabolic genes was varied, while the expression of GA signaling genes was not different between OE strain and WT (FIGS. 4A and 5). These results indicate that OsDREB2B is involved in GA metabolism associated with plant growth retardation, rather than GA signaling pathway. Among the GA metabolic genes, GA20ox and GA3ox convert GA precursors into bioactive gibberellins through a series of catalytic oxidation reactions, while GA2ox is considered to be a key factor in GA inactivation. The rice dwarf mutant d18 and the green revolution variety semi-dwarf 1 (sd 1) are respectively generated by the mutation of GA biosynthetic genes OsGA3ox2 and GA20ox 2. Mutations in GA20ox and GA3ox in maize (dwarf 1) and barley (sdw 1/denso) and overexpression of inactivated genes (OsGA 2ox1, osGA2ox6 and OsGA2ox 9) in rice resulted in moderate reduction in plant height by reducing GA content. These events occurred simultaneously in the inventors 'overexpressed OsDREB2B rice lines, similar to the inventors' previously identified AP2/ERF transcription factor OsRPH 1. The content of bioactive GA in the OE strain was significantly reduced compared to WT (P.ltoreq.0.01) (FIG. 4), while the content in mt was not significantly different from WT (FIG. 14). Furthermore, most of the bioactive GA precursors were reduced compared to WT, whereas in OE lines GA53 and some inactive catabolites were increased (fig. 4). The down-regulation of most GA20ox and GA3ox may lead to the accumulation of GA53, the initial precursor of GA1, while the up-regulation of GA2ox may lead to the increase of three catabolites, such as GA29, GA8 and GA51 (FIGS. 4 and 5).

The change of the GA metabolic gene expression quantity in the OE strain is consistent with the result of the reduction of the content of the bioactive GA, and shows that the GA biosynthetic gene is down-regulated and the GA inactivated gene is up-regulated. These results indicate that OsDREB2B, as a dual regulator of GA metabolic genes, negatively regulates the expression of GA synthesis genes, while positively regulating GA inactivation genes, results in a reduction in the content of bioactive gibberellins in rice.

AP2/ERF proteins bind by biological and abiotic stress response cis-acting elements in the promoter region of target genes, such as ethylene response elements (GCC-box, core motif: A/GCCGCC) and dehydration response elements/C-repeats (DRE/CRT, core motif: G/ACCGAC) (Allen et al, 1998). The DREB subgroup protein contains an AP2 conserved domain that specifically binds to the DRE/CRT cis-acting element of its regulatory gene (Shi et al, 2018). Overexpression of SlDREB, one of the DREB subgroup proteins in tomato, affects GA biosynthesis by down-regulating key genes for GA biosynthesis, thereby limiting internode elongation and leading to dwarfing. SlDREB acts as a direct repressor of SLCP in plants, probably by binding to the DRE/CRT element of SLCP (Li et al, 2012). Therefore, the inventors hypothesized that OsDREB2B may have similar functions to SlDREB. OsAP2-39 is an AP2/ERF protein belonging to the ERF subgroup and binds to GCC-box in the promoter of OsEUI, a gene of interest GA inactivation, an enzyme that catalyzes the 16 α,17 epoxidation of non-13-hydroxylated GAs, and has been shown to inactivate gibberellins in rice (Yaish et al, 2010). The OsDREB2B protein has transactivation activity and is localized to the nucleus (fig. 6). In the OE strain of the present inventors, the transcriptional expression of OsAP2-39 was up-regulated (A of FIG. 7), and OsDREB2B was directly bound to OsAP2-39 promoter (B-D of FIG. 7). OsDREB2B can specifically bind to DRE/CRT element (Chen et al, 2008), and the presence of DRE/CRT cis element in the promoter region of OsAP2-39 suggests that OsDREB2B directly regulates the transcript level expression of OsAP2-39 by binding to DRE/CRT element in its promoter. In this study, four GA-metabolizing genes had at least one GCC-box among the genes whose transcriptional expression was altered (FIG. 5), and 5 genes had DRE/CRT cis-elements in their promoter regions, indicating that OsDREB2B might directly regulate the gene containing DRE/CRT elements and indirectly regulate the gene containing GCC-box by binding to OsAP2-39 promoter.

Many AP2/ERF transcription factors regulate plant growth and development and stress responses through interaction with other transcription factors, such as BR regulation, WRKY, MYB and zinc finger transcription factors. In this study, osDREB2B physically interacted with WRKY transcription factor OsWRKY21 and was verified by Y2H analysis and BiFC analysis (fig. 8). OsWRKY21 overexpression rice shows dwarfing phenotype, and regulates the expression of GA metabolism and cell wall biosynthesis related genes. Interestingly, of the 15 genes whose expression was varied, 10 genes had at least one repeat of the W-box in their promoter regions. OsDREB2B and WRKY21 interact to regulate the expression of GA metabolic genes, and the genes contain W-box and/or DRE/CRT cis-elements in promoters thereof. In future research, osDREB2B overexpression rice taking Oswrky21 knockout mutant as background or/and OsWRKY21 overexpression rice taking OsDREB2B knockout mutation as background are/is created, and a molecular mechanism of protein interaction on plant growth and development is clarified.

The inventors propose an OsDREB2B model for controlling rice plant height by regulating the GA metabolic pathway (fig. 9). OsDREB2B upregulates GA-inactivated genes by interacting with OsWRKY21 either by direct binding to DRE/CRT-box or W-box elements, or by activating indirect binding of the OsAP2-39 gene to GCC-box. In contrast, the unidentified transcription repressing factor represses the binding of OsAP2-39, WRKY21/OsDREB2B complex to the promoter region of GA biosynthetic genes, resulting in transcription repression. Positive and negative regulation of GA metabolic genes results in a reduction in bioactive gibberellins, thereby reducing the plant height of rice.

In a word, the OsDREB2B is an AP2/ERF transcription factor, the content of bioactive GA is regulated by regulating GA biosynthesis genes, and GA inactivation genes are up-regulated in a mode of combining with an OsAP2-39 promoter and interacting with OsWRKY21, so that the height of rice plants is negatively regulated. The crosstalk mechanism of OsDREB2B in response to plant growth and stress will be further elucidated in future studies.

Claims

1. An AP2/ERF transcription factor OsDREB2B gene, the nucleotide sequence of which is shown in SEQ ID NO.1, is characterized by application in rice plant height regulation.

2. An overexpression vector of a rice AP2/ERF transcription factor OsDREB2B gene is characterized by comprising an OsDREB2B gene expression box containing a zea ubiquitin gene Ubi promoter, a target gene OsDREB2B, a 3' end untranslated region NOS of a nopaline synthetase coding region of agrobacterium, a screening marker gene expression box containing a cauliflower mosaic virus promoter CaMV35S, a marker gene HPT and a terminator NOS.

3. A recombinant cell containing the overexpression vector of the rice AP2/ERF transcription factor OsDREB2B gene of claim 2.